Our objective is to uncover the common mechanism by which Sec1p/Munc18-family (SM) proteins enhance SNARE assembly and membrane fusion. SM and SNARE proteins constitute the evolutionarily and structurally conserved core machinery for membrane fusion. Numerous mutations in SM and SNARE proteins have been identified that cause intellectual disability, epilepsy, immune disorders, and other disorders. In SNARE assembly, three or four SNAREs located on two membranes join into a parallel four- helix bundle like a zipper, forcing the membranes to fuse. However, SNAREs alone assemble slowly with limited specificity and accuracy. Despite extensive research in the past two decades, it remains unclear what mechanistic roles that SM proteins play in SNARE assembly, why they are essential for membrane fusion, and how they go wrong in diseases. Based on latest progresses, we hypothesize that SM proteins promote the rate and specificity of SNARE assembly, thereby enhancing membrane fusion. To test this hypothesis, we will examine several models. In particular, SM proteins bind SNARE motifs in register and act as a template to nucleate SNARE assembly. To test this and other models, we will measure the intermediates, energetics and kinetics of the coupled SNARE assembly and membrane fusion in the absence and presence of SM proteins. We will further elucidate the effects of disease-causing mutations in the process. Our primary tool is high-resolution optical tweezers combined with single-molecule fluorescence detection, which has successfully been applied to characterize assembly of cytoplasmic SNARE domains. Specifically, we will (1) measure the intermediates, energy, and kinetics of coupled trans-SNARE assembly and membrane fusion; (2) elucidate effects of SM proteins on trans-SNARE assembly and membrane fusion; (3) understand the molecular mechanisms of diseases or malfunctions caused by SNARE and SM mutations. Our research will reveal a common mechanism of SM-SNARE proteins and their pathologies.